Heart related problems remain a major public-health issue with high prevalence, poor clinical outcomes, and large health-care costs (Krum et al., 2009). +The major and ultimate result, heart failure, leads to significant morbidity and mortality. The primary causes of most syndromes contributing to heart failure include mainly hypertension, coronary artery disease, cardiomyopathy, infiltrative syndromes or inflammation results in a myocardium with a mixture of replacement fibrosis and diabetes. Though these conditions are treatable, they do not constitute a cure and only slow down the progression of the disease, which ultimately results in fatal heart failure (LaPointe et al., 2002). In recent times, the molecular pathways involved in induction and progression of most common cardiac diseases have been elucidated through advances in molecular cardiology and have led to the identification of numerous causative genes and proteins associated with these diseases. Gene therapy shows a promising means to control the production of such proteins to prevent and treat cardiac diseases (Lyon et al., 2008).
Gene therapy provides a possible alternative strategy to treat myocardial dysfunction (Fomicheva et al., 2008) whereby a therapeutic gene is delivered to the heart and expressed at high enough levels over a prescribed period of time to effect a therapeutic response (LaPointe et al., 2002). Among the heart-related conditions being targeted by gene therapy are genetic cardiomyopathies like Duchenne muscular dystrophy (DMD) (Bostick et al., 2009), hypertrophic cardiomyopathy (Jacques et al., 2008), and diabetic cardiomyopathy (Wang et al., 2009). Over the past decade, gene therapy has shown promising results in preclinical studies in animal models to treat heart failure including arrhythmia, restenosis (Müller et al., 2007), ischaemia and hypoxia (Fomicheva et al., 2008). For instance, therapeutic angiogenesis has been explored by gene therapy to treat cardiac ischemia by over-expressing genes encoding angiogenic factors (e.g., vascular endothelial growth factor or VEGF (Müller et al., 2007; Stewart et al., 2009). In addition, gene therapy provides a potential strategy to create an immune privileged site within the transplanted heart to prevent immune rejection in transplant patients (Vassalli et al., 2009). Clinical successes of gene therapy to treat cardiac diseases have been slower than originally predicted, due to challenges inherent to gene transfer efficacy: inadequate delivery to the target tissue, loss of therapeutic effect, and dose-limiting interactions with the host immune system (Sasano et al., 2007). Delivery issues are the most challenging among these. Reported methods of myocardial delivery include intramyocardial injection, coronary catheterization, pericardial delivery, ventricular cavity infusion during aortic cross-clamping, and perfusion during cardiopulmonary bypass (Müller et al., 2007; Sasano et al., 2007).
The use of plasmid/naked DNA has been demonstrated to yield therapeutic effects in animal models and patients with intractable myocardial ischemia. But since naked DNA cannot enter cells spontaneously with sufficient efficiency, systemic injection of naked DNA is an inefficient technique for myocardial gene delivery. Though the transfection efficiency can typically be enhanced if the naked DNA is coupled to compounds like liposomes, cholesterol-lipopolymers, poloaxime nanospheres and gelatin, these compounds do not enhance myocardial specificity (Lyon et al., 2008). Hence, naked DNA delivery to the heart must be carried out by direct intramyocardial injection (Müller et al., 2007) or by sonoporation or (UTMD) ultrasound targeted microbubble destruction, although transfection efficiency remains limiting (Dishart et al., 2003; Lyon et al., 2008).
Even though non-viral vectors and plasmid DNA are safe and relatively low cost, they only lead to transient transfection since they are unable to integrate into the host genome or persist in episomal forms. This makes them unsuitable for long-term gene expression as required in heart failure or hereditary cardiac diseases like cardiomyopathies, but they may be suitable for applications involving angiogenesis which is transient (Müller et al., 2007). These show that plasmid DNA is inefficient at myocardial gene delivery and expression (Lyon et al., 2008).
Four classes of viral vectors have predominantly been used for myocardial gene delivery: retroviral, lentiviral, adenoviral (Ad) and AAV vectors (Lyon et al., 2008). Among these, AAV has been proven to be more efficient compared to other vectors to transduce the heart. Adenoviral vectors have also been shown to transduce the heart whereas myocardial transduction with retroviral or lentiviral vectors is relatively inefficient. Most investigators have switched to AAV to achieve long-term expression and overcome inflammatory characteristics inherent to Ad (Sasano et al., 2007). No obvious pathology has been observed in connection with AAV (Gödecke et al., 2006) with consistent sustained expression of gene delivered by AAV vector for several months (Lyon et al., 2008).
AAV vectors allow long-term gene transfer to the heart in animal models, and skeletal muscle in humans but the specificity can be increased by transcriptional targeting (Goehringer et al., 2009). In the heart, AAV2 vectors utilize specific cell surface receptors including heparin sulphate proteoglycans, human fibroblast growth factor receptor 1, and integrins αvβ5/α5β1 to enter the cells via receptor-mediated endocytosis. They exploit the transcytosis trafficking pathway of endothelial cells, in order to cross the endothelial barrier to reach cardiomyocytes after intravascular delivery. The recombinant AAVs (rAAV), which are used for gene therapy do not integrate into the genome of cardiomyocyte but rather exist as episomal DNA (Lyon et al., 2008).
Among the 12 serotype classes of AAV currently identified, AAV1, AAV6, AAV8 and AAV9 have been observed to have higher tropism for myocardium than all the alternative candidate vectors for cardiac gene therapy. Also among these four, AAV9 was the most efficient vector for cardiac gene delivery (VandenDriessche et al., 2007). In mouse models, a 220-fold increase in myocardial transduction efficiency was obtained after only a single intravenous dose of AAV9 carrying a reporter gene was administered compared to the relatively cardiotropic AAV1 (Lyon et al., 2008).
Despite the success of using rAAV vectors for gene therapy, some obstacles still remain. The problem of neutralizing antibodies directed against the vector capsid is one major obstacle, which prevents re-administration of AAV vectors of the same serotype (Kwon et al., 2008). Moreover, T cell immune responses directed against the AAV capsid antigens presented in association with major histocompatibility complex class I antigens (MHC-I) on the surface of transduced target cells may curtail long-term gene expression. Other problems include limited tissue tropism for serotypes that bind heparan sulfate; poor infection of refractory cell types, such as stem cells; challenges with high-efficiency targeted gene delivery to specific cell populations (VandenDriessche et al., 2007); relatively restricted packaging capacity and inefficient large-scale production (Lyon et al., 2008); a relatively slow onset of gene expression, possibly owing to cytoplasmic trafficking, vector uncoating and conversion of the single-stranded genome into double-stranded DNA (Müller et al., 2007; Douar et al., 2001). A particular problem is achieving high expression levels that remain cardiac specific. While this can be done through the use of cardiac-specific promoters, drawbacks of these may include the large size of these promoters, since many vectors have a restricted cloning space, and/or the low activity compared to strong (viral) promoters, such as cytomegalovirus (CMV) or long teiniinal repeat (LTR) promoter sequences, widely used in gene therapy protocols.
To avoid transduction of other tissues, for instance skeletal muscle or liver (Yue et al., 2008, VandenDriessche et al., 2007), transcriptional targeting may be employed to increase the specificity of the vector for cardiac specific experimentation (Goehringer et al., 2009). Tissue specific promoters retain specificity and so are good promoters for viral vectors (Reynolds et al., 2001) since non-specific promoters can potentially elicit immune response against the vector plasmid (Cao et al., 2002) especially for intravenous vector application (Goehringer et al., 2009). It is also noted that tissue-specific promoters may also be considered inducible promoters because they may be induced by endogenous or exogenous factors as well (Venter et al., 2007). Many promoters have been used in gene therapy investigations for cardiac delivery, such as human cytomegalovirus (CMV) promoter (Fomicheva et al., 2008; Phillips et al., 2002), muscle creatine kinase (MCK) promoter, myosin light chain (MLC) 2v promoter (Gruber et al., 2004; Su et al., 2004), alpha myosin heavy chain promoter (Bostick et al., 2009; Black et al., 2002; Aikawa et al., 2002). Alpha myosin heavy chain (αMHC) promoter has been the most commonly used myocardial-specific promoter (Buerger et al., 2006) and has shown highly specific and robust levels of expression in the heart in most studies. The (MLC) 2v promoter has also been used by various cardiac gene therapy researchers but has been found to have a partial expression in the liver (Phillips et al., 2002; Su et al., 2004). Comparative study on five different tissue specific promoters in AAV9 vector has been conducted by Pacak et al., and the comparative analysis revealed that αMHC promoter confer the most cardiac specific expression (Pacak et al., 2008). The choice of promoters that has been used in the context of gene therapy vectors nevertheless remains relatively limited. Moreover, the robustness of many currently used heart specific promoters can still be augmented.
Increasing tissue-specific transgene expression is desirable as a way to decrease the amount of viral vector required to achieve a clinical effect. To increase activity, the use of cis-acting regulatory elements has been proposed. Typically, this concerns enhancer sequences, i.e., nucleic acid sequences that increase a promoter's activity and that have the potential to act in cis, and regardless of their orientation, even over relatively long distances (up to several kilobases away from the target promoter). However, enhancer function is not necessarily restricted to such long distances as they may also function in close proximity to a given promoter. Some enhancers like CMV, αMHC, Rous sarcoma virus genome long terminal repeats (RSV), Simian virus (SV40), human phosphoglycerate kinase (PGK), and the muscle creatine kinase (MCK) are widely used in various vectors and transgenic animals (Xu et al., 2001; Wang et al., 2008; Salva et al., 2007). Most researchers have made use of the human CMV immediate-early enhancer to express a transgene (Xu et al., 2001; Gruh et al., 2008; Müller et al., 2000; Raake et al., 2008).
Sometimes, chimeric enhancer/promoters are constructed by swapping enhancer/promoter units from different sources for a greater effect. The most widely used of such a chimera is the CAG-promoter which results from a combination of CMV immediate-early enhancer, a chicken β-actin gene promoter and a rabbit β-globin splice acceptor or intron, making it drive strong gene expression in several tissues via viral or non-viral vectors (Xu et al., 2001; Wang et al., 2008). However, such enhancers do not increase specificity as they are not restricted to cardiac tissue.
To be able to provide a therapeutic level of the transgene product for an extended time period, gene transfer vectors preferably allow specifically regulated, high expression, while at the same time retaining sufficient cloning space for the transgene to be inserted, i.e., the regulatory elements used to achieve the high and tissue-specific expression preferably are of only limited length. However, none of the gene therapy vectors disclosed thus far satisfies all these criteria. Instead, gene therapy vectors are not sufficiently robust in terms of either expression levels and/or specificity of expression in the desired target cells, particularly cardiac cells. Decreasing the promoter/enhancer size often compromised the expression levels and/or expression specificity whereas the use of larger sequences often compromises the efficiency of gene delivery due to impaired vector function, packaging and/or transfection/transduction efficiency. Thus, there is a need in the art for vectors that achieve therapeutic levels of transgene expression in the heart for effective gene therapy.